Thermocellulases for lignocellulosic degradation

ABSTRACT

Thermostable cellulase enzyme systems comprising at least one each of a thermostable endoglucanase, an exo-processive-endoglucanase, and a β-glucosidase carry out the complete, coordinated hydrolysis of crystalline cellulose to monomeric glucose.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of issued U.S. Pat. No. 8,847,031 B2, issued Sep. 30, 2014, and claims the benefit of U.S. Provisional Patent Application Ser. No. 61/079,208 filed on Jul. 9, 2008 and international patent application No. PCT/US2009/050080, filed Jul. 9, 2009, and incorporates said applications by reference into this document as if fully set out at this point.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with Government support under Grant No. DE-FG36-06GO16107, awarded by the U.S. Department of Energy. The Government may have certain rights in this invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention generally relates to thermostable enzymes capable of degrading (hydrolyzing) cellulose at high temperatures, and the incorporation of nucleic acids coding for one or more of such enzymes into a host, and, more particularly, a host that produces or is composed of cellulosic material.

2. Background of the Invention

Cellulose is a polysaccharide consisting of a linear chain of several hundred to over nine thousand β (1→4) linked D-glucose units [formula (C₆H₁₀O₅)n]. Cellulose is the most abundant organic compound on earth, making up about 33 percent of all plant matter, about 50 percent of wood, and about 90 percent of products such as cotton. In nature, cellulose is present as part of the lignocellulosic biomass of plants, which is composed of cellulose, hemicellulose, and lignin. The carbohydrate polymers (cellulose and hemicelluloses) are tightly bound to the lignin, by hydrogen and covalent bonds.

Many highly desirable products are derived from lignocellulosic biomass. In particular, much interest has recently been focused on recapturing the saccharide building blocks locked in plant biomass for biofuel production. For example, fermentation of plant biomass to ethanol is an attractive carbon neutral energy option since the combustion of ethanol from biomass produces no net carbon dioxide in the earth's atmosphere. Further, biomass is readily available, and its fermentation provides an attractive way to dispose of many industrial and agricultural waste products. Finally, plant biomass is a highly renewable resource. Many dedicated energy crops can provide high energy biomass, which may be harvested multiple times each year.

One barrier to the production of products from biomass is that the cellulosic polymer has evolved to resist degradation and to confer hydrolytic stability and structural robustness to the cell walls of plants. This robustness or “recalcitrance” is due largely to extensive intermolecular hydrogen bonding between cellulose polymer chains. Some organisms, notably fungi, bacteria, and protozoans, but also some plants and animals, have evolved the ability to digest cellulose. In vivo cellulose breakdown typically entails the cooperative interaction of several cellulases, enzymes that catalyze the cellulolysis (hydrolysis) of cellulose. Several different kinds of cellulases, which differ structurally and mechanistically, are known, and some of these have been isolated, characterized and used to break down cellulose in vitro. General categories of cellulases include: endo-cellulases (endoglucanases), which randomly hydrolyze internal bonds to disrupt the crystalline structure of cellulose, thereby exposing individual cellulose polysaccharide chains; and exo-cellulases (exo-processive-endoglucanases), which cleave 2-4 units from the ends of the exposed chains produced by endocellulases to produce tetrasaccharides or disaccharides such as cellobiose. Two major types of exo-cellulases are known, one of which works processively from the reducing end, and one of which works processively from the non-reducing end of cellulose. A third major type of cellulase is cellobiase or beta-glucosidase, which hydrolyses exo-cellulase products such as cellobiose into individual glucose monosaccharides.

Typically, the digestion of cellulose is carried out at temperatures approaching 100° C. because, at high temperatures, intermolecular hydrogen bonds are disrupted and recalcitrant cellulose polymers become accessible to the cellulase enzymes. Therefore, cellulases used commercially in such processes must be able to withstand very high temperatures, preferably for extended periods of time.

There is an ongoing need to identify, isolate and characterize cellulases, especially thermally stable cellulases, for use in the enzymatic hydrolysis of cellulose. Of particular interest is the development of groups or systems of cellulases that include enzymes with endo-cellulase, exo-cellulase and beta-glucosidase activity, the enzymes in the system acting in concert to carry out the complete hydrolysis of cellulose to glucose at high temperatures.

SUMMARY OF THE INVENTION

Protein sequences which heretofore were not recognized as having enzymatic activity have been isolated and characterized as thermostable enzymes capable of degrading (hydrolyzing) cellulose at high temperatures. The activity is referred to herein as cellulase or cellulase-like. The enzymes, originating from Archaea and various thermophilic bacteria, include: endoglucanases that randomly hydrolyze internal glycosidic bonds; exo-processive-endoglucanases that split off cellobiose dimers; and β-glucosidases that reduce cellobiose into monomeric glucose molecules. While the β-glucosidase enzymes are technically not “cellulases” because cellobiose (not cellulose) is the substrate they cleave, the three groups of enzymes may be sometimes collectively referred to as “cellulases” herein. The enzymes are optimally catalytically active at temperatures at or above about 85° C. and retain >85% of their enzymatic activity even after a 5 day incubation at elevated temperature, e.g. 90° C. In some embodiments, the enzymes, or enzyme systems or groupings comprising multiple thermostable catalytic activities may advantageously be used to degrade cellulose. Preferably, in the case of systems which have multiple thermostable catalytic activities, such a system comprises at least one endoglucanase, at least one exo processive-endoglucanase, and at least one beta-glucosidase enzyme, and thus can carry out the complete hydrolysis of cellulose to glucose at high temperatures in a sequential, cooperative manner. Catalytic consolidation at high-temperatures using the enzyme systems described herein is not additive but synergistic, accessing recalcitrant cellulose and hydrolyzing beta linkages at temperatures above 85° C. Thus, one aspect of the invention is to employ the enzymes, alone or in a group, in processes to break down cellulosic material by contacting the cellulosic material with the enzymes and elevating the temperature to activate the enzymes to break down the cellulosic material. These processes might be performed, for example, in tanks where the cellulosic material is distributed in a liquid carrier; however, the enzymatic breakdown may be achieved simply through elevating the temperature of the cellulosic material with the enzymes being in contact with the cellulosic material.

The invention also contemplates the incorporation of nucleic acids coding for one or more of the enzymes into a host (e.g., a plant, fungi, bacterium or animal). In the case where the host produces or is composed of cellulosic material (e.g., plants such as corn, switch grass, sugar cane, sorghum, pinus and eucalyptus), the host can be subjected to breakdown of the cellulosic material, for example, after harvest. That is, in a particular example, corn or switchgass transformed to include nucleic acids coding for the enzymes will express the enzymes internally, and after collection or harvest of the corn or switchgrass, the enzymes can be activated to begin and preferably ultimately to completely degrade the cellulose simply by elevating the temperature of the corn or switchgrass.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-B. Pyrococcus furiosus Termocel 1 endoglucanase. A, nucleotide sequence (903 bp, SEQ ID NO: 1); and B, amino acid sequence (301 aa, SEQ ID NO: 2). Nucleotide sequence is optimized for expression in corn and shown without N-terminal signal peptide encoding sequence.

FIG. 2A-B. Thermotoga petrophila Termocel 2 endoglucanase. A, nucleotide sequence (825 bp, SEQ ID NO: 3); and B, amino acid sequence (274 aa, SEQ ID NO: 4).

FIG. 3A-B. Pyrococcus horikoshii Termocel 3 exocellulase. A, nucleotide sequence (1377 bp, SEQ ID NO: 5); and B, amino acid sequence (458 aa, SEQ ID NO: 6).

FIG. 4A-B. Pyrococcus abyssi Termocel 4 exocellulase. A, nucleotide sequence (1542 bp, SEQ ID NO: 7); and B, amino acid sequence (514 aa, SEQ ID NO: 8). Nucleotide sequence is optimized for expression in rice and shown without stop codon.

FIG. 5A-B. Thermotoga petrophila Termocel 5 endoglucanase. A, nucleotide sequence (987 bp, SEQ ID NO: 9); and B, amino acid sequence (328 aa, SEQ ID NO: 10).

FIG. 6A-B. Caldivirga inaquilingenesis Termocel 6 endoglucanase. A, nucleotide sequence (852 bp, SEQ ID NO: 11); and B, amino acid sequence (284 aa, SEQ ID NO: 12). Nucleotide sequence is optimized for expression in corn.

FIG. 7A-B. Thermotoga petrophila Termocel 7 beta-glucosidase. A, nucleotide sequence (2169 bp, SEQ ID NO: 13); and B, amino acid sequence (722 aa, SEQ ID NO: 14).

FIG. 8A-B. Thermotoga petrophila Termocel 8 beta-glucosidase. A, nucleotide sequence (1341 bp, SEQ ID NO: 15); and B, amino acid sequence (446 aa, SEQ ID NO: 16).

FIG. 9. Characteristics of high-temperature operating thermo-stable cellulases. swAvicel=phosphoric acid swollen Avicel; _(av)Cellulose=Avicel; cellulase specific activity is expressed as μM of reducing sugar/mg protein/day at 85° C., pH 6; beta-glucosidase specific activity is expressed as nM p-nitrophenol (pN)/μg protein/minute.

FIG. 10A-E. Processive exocellulases (cellobiohydrolase). Capillary zone electrophoresis (CZE) of 8-aminonaphthalene-1,3,6 trisulfonic acid (ANTS)-labeled cellopentose breakdown products incubated with Termocel 1 (A), Termocel 2 (B) and Termocel 3 (C). CZE retention times (D) of purified monomer, (DPI) dimer (DP2), trimer (DP3) and the substrate cellopentose (DP5). Sequential predicted cleavage pattern (E) between DP5 and DP4, DP4 and DP3, DP3 and DP2. Assay conditions, Substrate, ANTS-cellopentose (FIG. 9C), buffer sodium phosphate/citrate 50 mM, incubated at 95° C., pH 6.

FIG. 11A-E. Processive endocellulases (endoglucanase). Capillary zone electrophoresis (CZE) of ANTS-labeled cellopentose breakdown products incubated with Termocel 4 (A), Termocel 5 (B) and Termocel 6 (C). CZE retention times (D) of purified monomer, (DPI) dimer (DP2), trimer (DP3) and the substrate cellopentose (DP5). Predicted cleavage pattern (E) between DP2 ad DP3 and DP3 and DP4. Assay conditions, Substrate, ANTS-cellopentose (FIG. 9C), buffer sodium phosphate 50 mM, incubated at 95° C., pH 6.

FIG. 12. Temperature optima for high-temperature catalytic cellulases, and activities at 60, 45 and 20° C. _(sw)Avicel=phosphoric acid swollen Avicel; _(av)Cellulose=Avicel; cellulase specific activity is expressed as μM of reducing sugar/mg protein/day at 85° C., pH 6; beta-glucosidase specific activity is expressed as nM p-nitrophenol (pN)/μg protein/minute.

FIG. 13A-D. Termocel thermostability. Termocels were incubated at 90° C. in phosphate/citrate buffer for the indicated number of hours and CMC or PNPG activity determined. The amount of residual activity is shown. A, Termocels 1 and 2; B, Termocels 3 and 4; C, Termocels 5 and 6; D, Termocels 7 and 8 With the exception of Termocel 3, all cellulases retained >80% of activity after 120 hrs at 90° C. Thus, these enzymes are stable at high temperatures.

FIG. 14. Flow chart illustrating cellulose treatment steps.

FIG. 15. Table depicting biomass substrate specificity of particular Termocels.

FIG. 16A-B. Caldivirga maquilingensis Termocel 9 endoglucanase. A, nucleotide sequence (1230 bp, SEQ ID NO: 29); and B, amino acid sequence (410 aa, SEQ ID NO: 30). Nucleotide sequence is optimized for expression in corn.

FIG. 17A-B. Pyrococcus horikoshii Termocel 10 endoglucanase. A, nucleotide sequence (2214 bp, SEQ ID NO: 31); and B, amino acid sequence (737 aa, SEQ ID NO: 32).

FIG. 18A-F. Nucleotide sequences as set forth in SEQ ID NO: 1 (from Pyrococcus furiosus), SEQ ID NO: 3 (from Thermotoga petrophila), SEQ ID NO: 5 (from Pyrococcus horikoshii), SEQ ID NO: 7 (from Pyrococcus abyssi), SEQ ID NO: 9 (from Thermotoga petrophila), SEQ ID NO: 11 (from Caldivirga maquilingenesis), SEQ ID NO: 29 (from Caldivirga maquilingensis), SEQ ID NO: 31 (from Pyrococcus horikoshii), SEQ ID NO: 13 (from Thermotoga petrophila), and SEQ ID NO: 15 (from Thermotoga petrophila).

FIG. 19A-C. Amino acid sequences as set forth in SEQ ID NO: 2 (from Pyrococcus furiosus), SEQ ID NO: 4 (from Thermotoga petrophila), SEQ ID NO: 6 (from Pyrococcus horikoshii), SEQ ID NO: 8 (from Pyrococcus abyssi), SEQ ID NO: 10 (from Thermotoga petrophila), SEQ ID NO: 12 (from Caldivirga maquilingenesis), SEQ ID NO: 30 (from Caldivirga maquilingensis), SEQ ID NO: 32 (from Pyrococcus horikoshii), SEQ ID NO: 14 (from Thermotoga petrophila), and SEQ ID NO: 16 (from Thermotoga petrophila).

DETAILED DESCRIPTION

The present invention is based on the identification and characterization of a comprehensive set of thermostable cellulases that work in concert to catalyze the hydrolysis of cellulose to glucose at very high temperatures. The cellulases, originally identified in archeal and bacterial genomes, include endoglucanases that randomly cleave internal glycosidic bonds; exo-processive-endoglucanases that further hydrolyze cellulose fragments into cellobiose dimers; and β-glucosidases that further hydrolyze cellobiose dimers to glucose monomers. While the enzymes may be used individually, in some embodiments of the invention they are grouped to form a cooperative enzyme system. By combining into a group at least one endoglucanase, at least one exo-processive-endoglucanase and at least one β-glucosidase, an enzyme system is formed that is capable of the complete breakdown of cellulose to glucose. Importantly, the enzyme system's various catalytic activities are optimal at temperatures that are high enough to destabilize the hydrogen bonds between crystalline cellulose strands (e.g. at temperatures greater than 80° C.). Destabilization of hydrogen bonds at high temperatures causes disruption of the crystalline structure of cellulose, thereby facilitating access by the first enzyme in the series (endoglucanase) to internal glycosidic bonds of individual cellulose polymer strands, and allowing the step-wise process of cellulose breakdown to begin.

Exemplary amino acid sequences of the recombinant enzymes of the invention and exemplary nucleotide sequences that encode them are depicted in FIGS. 1-8. However, those of skill in the art will recognize that the invention also encompasses variant proteins comprising amino acid sequences that are based on or derived from the sequences disclosed herein. By an amino acid sequence that is “derived from” or “based on” the sequence disclosed herein, we mean that a derived sequence (or variant sequence) displays at least about 50 to 100% identity to an amino acid sequence disclosed herein, or about 60 to 100% identify, or about 70 to 100% identity, or even from about 80 to 100% identify. In preferred embodiments, a variant sequence displays from about 90 to 100% or about 95 to 100% amino acid identity. In further preferred embodiments, a variant sequence is 95, 96, 97, 98 or 99% identical to at least one sequence disclosed herein. Variations in the sequences may be due to a number of factors and may include, for example: conservative or non-conservative amino acid substitutions; natural variations among different populations as isolated from natural sources; various deletions or insertions (which may be amino terminal, carboxyl terminal, or internal); addition of leader sequences to promote secretion from the cell; addition of targeting sequences to direct the intracellular destination of a polypeptide; etc. Such alterations may be naturally occurring or may be intentionally introduced (e.g. via genetic engineering) for any of a wide variety of reasons, e.g. in order to eliminate or introduce protease cleavage sites, to eliminate or introduce glycosylation sites, in order to improve solubility of the polypeptide, to facilitate polypeptide isolation (e.g. introduction of a histidine or other tag), as a result of a purposeful change in the nucleic acid sequence (see discussion of the nucleic acid sequence below) which results in a non-silent change in one or more codons and thus the translated amino acid, in order to improve thermal stability of the protein, etc. All such variant sequences are encompassed by the present invention, so long as the resulting polypeptide is capable of catalyzing the enzyme activity of the original protein as disclosed herein. For example, the invention includes shorter portions of the sequences that also retain the catalytic activity of the enzyme. The full-length protein sequences and/or active portions thereof are both referred to as polypeptides herein. In addition, the invention also includes chimeric or fusion proteins that include, for example: more than one of the enzymes disclosed herein (or active portions thereof); or one or more of the enzymes disclosed herein (or portions thereof) plus some other useful protein or peptide sequence(s), e.g. signal sequences, spacer or linker sequences, etc.

The invention also comprehends nucleic acid sequences that encode the proteins and polypeptides of the invention. Several exemplary nucleic acid sequences are provided herein. However, as is well known, due to the degeneracy of the nucleic acid triplet code, many other nucleic acid sequences that would encode an identical polypeptide could also be designed, and the invention also encompasses such nucleic acid sequences. Further, as described above, many useful variant forms of the proteins and peptides of the invention also exist, and nucleic acid sequences encoding such variants are intended to be encompassed by the present invention. In addition, such nucleic acid sequences may be varied for any of a variety of reasons, for example, to facilitate cloning, to facilitate transfer of a clone from one construct to another, to increase transcription or translation in a particular host cell (e.g. the sequences may be optimized for expression in, for example, corn, rice, yeast or other hosts), to add or replace promoter sequences, to add or eliminate a restriction cleavage site, etc. In addition, all genera of nucleic acids (e.g. DNA, RNA, various composite and hybrid nucleic acids, etc.) encoding proteins of the invention (or active portions thereof) are intended to be encompassed by the invention.

The invention further comprehends vectors, which contain nucleic acid sequences encoding the polypeptides of the invention. Those of skill in the art are familiar with the many types of vectors, which can be useful for such a purpose, for example: plasmids, cosmids, various expression vectors, viral vectors, etc.

Production of the nucleic acids and proteins of the invention can be accomplished in any of many ways that are known to those of skill in the art. The sequences may be synthesized chemically using methods that are well-known to those of skill in the art. Alternatively, nucleotide sequences may be cloned using, for example, polymerase chain reaction (PCR) and/or other known molecular biology and genetic engineering techniques Recombinant proteins may be made from a plasmid contained within a bacterial host such as Escherichia coli, in insect expression systems, yeast expression systems, plant cell expression systems, etc. Further, the nucleic acid sequences may be optimized for expression in a particular organism or system. To that end, the present invention also encompasses a host cell that has been transformed or otherwise manipulated to contain nucleic acids encoding the proteins and polypeptides of the invention, either as extra-chromosomal elements, or incorporated into the chromosome of the host. In particular, in the practice of the present invention, nucleic acid sequences encoding one or more of the cellulases (e.g. an entire “system” as described herein) may be introduced into plant cells, seeds, etc., to generate recombinant plants that contain the nucleic acids.

Plant transformation to incorporate one or more nucleic acids coding for one or more cellulase enzymes described herein can be accomplished by a variety of techniques known to those of skill in the art. Plant transformation is the introduction of a foreign piece of DNA, conferring a specific trait, into host plant tissue. Plant transformation can be carried out in a number of different ways; Agrobacterium mediated transformation, particle bombardment, electroporation and viral transformation.

Suitable examples of plants that may be transformed to include one or more cellulase enzymes or sets of enzymes include but are not limited to rice, corn, various grasses such as switchgrass, sugar cane, sorghum, pinus and eucalyptus, etc. Advantages of genetically engineering plants to contain and express the cellulase genes include but are not limited to the availability of the enzymes within the cell wall tissues (cellulosic fibers) and ready to be activated by high temperatures (e.g., heating to 70 or 80 C or more). Deposition of these enzymes produced by the plant cells and targeted to the apoplast, should largely overcome the recalcitrant nature of biomass.

The cellulases and/or cellulase enzyme systems of the invention may be used for the breakdown (catalysis) of cellulose in biomass from a wide variety of sources. Biomass comes in many different types, which may be grouped into four main categories: (1) wood residues (including sawmill and paper mill discards); (2) municipal paper waste; (3) agricultural residues (including corn stover and sugarcane bagasse); and (4) dedicated energy crops, which are mostly composed of fast growing tall, woody grasses. Cellulose-containing biomass from any of these or other sources may be acted upon by the enzymes and consolidated enzyme systems of the invention.

Generally, the breakdown of cellulose will be complete, i.e. the endproduct is glucose. This is especially true when a consolidated enzyme system that includes at least three different types of enzymes (for example, an endoglucanase, an exo-processive-endoglucanases, and a β-glucosidase) are employed. However, this need not always be the case. Depending on the goal of the reaction, only one enzyme may be utilized (e.g. an endoglucanase to generate randomly cleaved cellulose polymers); or only two enzymes may be utilized (e.g. an endoglucanase and an exo-processive-endoglucanases to generate dimeric disaccharides such as cellobiose), etc. Any desired grouping of the enzymes of the invention may be utilized to generate any desired endproduct that the enzymes are capable of producing from a suitable substrate. Further, one or more of the enzymes of the invention may be used in combination with other cellulases, or with enzymes having other types of activities. In one embodiment of the invention, a “system” could further include a yeast or other organism capable of fermenting glucose to e.g. ethanol.

The cellulases of the invention have very high temperature optima, an optimal temperature being the temperature at which an enzyme is maximally active (e.g. as an endoglucanase, an exo-processive-endoglucanases, or a β-glucosidase), as determined by a standard assay recognized by those of skill in the art. As described in the Examples section below, the lowest temperature optimum for an enzyme of the invention is about 85° C., and the highest temperature optimum is about 102° C. Further, the enzymes of the invention are thermally stable, i.e. they are capable of retaining catalytic activity at high temperatures (e.g. at their temperature maximum, or at temperatures that deviate somewhat from the maximum) for extended periods of time, for example, for at least for several hours (e.g. 1-24 hours), and in many cases, for several days (e.g. from 1-7 days or even longer). By “retain catalytic activity” we mean that the enzyme retains at least about 10, 20, 30, 40 or 50% or more of the activity displayed at the beginning of the extended time period, when measured under standard conditions; and preferably the enzyme retains 60, 65, 70, 75, 80, 85, 90, 95, or even 100% of the activity displayed at the beginning of the extended time period.

The enzymes of the invention are generally employed in reactions that are carried out at temperatures at or near those which are optimal for their activity. Some enzymes may be used over a wide temperature range (e.g. at a temperature that is about 50, 40, 30, 20, 10, 5 or fewer degrees lower than (below) the temperature optimum, and up to about 5, 10, 15, or more degrees greater than (above) the temperature optimum. For other enzymes, the range may be more restricted, i.e. they may display catalytic activity within a narrow temperature range of only less than about 10, or less than about 5, or fewer degrees of their optimal catalytic temperature. When carrying out a cellulose digestion reaction, the enzymes may be used one at a time sequentially (i.e. one enzyme is added, reaction occurs, and then another enzyme is added, with or without removal of the previous enzyme, and so on), or the reaction mixture may contain two or even all three of the enzymes (an enzyme system) may be added at the same time. When designing groups of enzymes to be included in an enzyme system, those of skill in the art will recognize that a suitable temperature at which all enzymes in the group are active will be selected as the temperature for reaction. Or, conversely, if it is desired to carry out a reaction at a particular temperature, enzymes with optimal activity at or near that temperature would be selected for inclusion in the set. For example, for a reaction to be carried out at 97° C., one might choose a set of enzymes that includes Termocel 5 (endocellulase, optimum=96° C.), plus Termocel 2 (exocellulase, optimum=98° C.), plus Termocel 7 (β-glucosidase, optimum=98° C.); whereas for a reaction that is to be carried out at 90° C., one might choose Termocel 6 (endocellulase, optimum=85° C.), plus Termocel 3 (exocellulase, optimum=94° C.), plus Termocel 7 (β-glucosidase, optimum=92° C.). If an enzyme is used individually, the reaction may be carried out at a temperature near its optimum, or at which the enzyme retains sufficient activity to be useful. In addition, the selection of a reaction temperature may be based on other considerations, e.g. safety or other practical considerations of high temperature operations, or concerns about the cost of keeping a reaction mixture at a high temperature, the temperature used for preparing biomass for the reaction, the temperature of procedures that follow the reaction, etc. Generally, the degradation of cellulose will be carried out at a temperature in the range of from about 70 to about 95° C.

The invention also provides methods of use of the enzymes disclosed herein. The methods generally involve the used of at least three enzymes of the invention, at least one from each of the three classes endoglucanase, exo-processive-endoglucanases, and β-glucosidase. The three classes of enzymes act in concert to sequentially breakdown cellulose to glucose. The methods of the invention may be carried out for any purpose for which it is desirable to prepare glucose (or other products produced by the enzymes), and further metabolize into other chemicals, such as ethanol, xylitol, butanol, amino acids, glycol etc.

Generally, such methods are carried out by first pretreating a cellulose-rich feedstock by removing the lignin (usually through ball milling). The production of sugars (saccharification) of the pretreated cellulose is carried out by suspending the pretreated cellulose in a cellulase broth that contains suitable cellulase enzymes such as those disclosed herein. Generally, the reaction will be carried out at a temperature in the range of from about 70 to about 95 C, and the length of time for a reaction will be in the range of from about one hour to about six days. Reactions are carried out in media such as aqueous buffered to a suitable pH, e.g. in the range of from about pH 4 to about pH 9.

Thereafter, the desired products (e.g. glucose and cellobiose) may be harvested from the broth, or the reaction products may be further processed. For example, for the production of ethanol, fermentation of the glucose in the broth may be carried out by known conventional batch or continuous fermentation processes, usually using yeast. Ethanol may be recovered by known stripping or extractive distillation processes. This process is illustrated schematically in FIG. 14, which shows the steps of pretreating biomass to provide a source of cellulose; contacting the cellulose with one or more cellulase enzymes of the invention to hydrolyze cellulose to glucose, and fermenting the glucose to produce ethanol.

EXAMPLES Example 1 Isolation and Characterization of Cellulases that Catalyze High-Temperature Thermo-Stable Bio-Consolidated Cellulose Breakdown Abstract

Cellulose breakdown entails cooperative interaction of various cellulases by accessing and cleaving the recalcitrant cellulosic polymer. At high temperatures, most of the recalcitrant biomass polymers become enzymatically accessible because of intermolecular hydrogen bond disruption. Here, we describe a high-temperature operating thermo-stable cellulose enzyme system, consisting of endoglucanases, exoprocessive-endoglucanases and beta-glucosidases. Two catalytic types of cellulose cleaving enzymes was found: endoglucanases that randomly hydrolyze internal glycosidic bonds and exo-processive-endoglucanase, which split off cellobiose dimers. Finally, a third activity, βglucosidase, reduces cellobiose into glucose molecules. The consolidated enzyme system operates optimally at temperatures above 85° C. and retains >85% of its enzymatic activity after a 5 day incubation at 90° C. Catalytic consolidation with high-temperatures is not additive but synergistic, accessing recalcitrant cellulose and hydrolyzing beta linkages above 85° C.

Introduction

Cellulose is an abundant biopolymer component of plant cell walls. Cellulose is a linear biopolymer of D-glucose, linked by β-1,4-glucosyl linkages. Cellulosic enzyme systems completely hydrolyze cellulose rendering glucose molecules. A cellulosic enzymatic system consists of multiple cellulases, endo-β-glucanase, cellobiohydrolase and β-glucosidase, which interact synergistically in producing glucose. Endoglucanases randomly hydrolyze the internal glycosidic bonds to decrease the length of the cellulose chain. Cellobiohydrolases are exo- or endo-processive enzymes that split off cellobiose of the shortened cellulose chains. Cellobiose is hydrolyzed by β-glucosidase to glucose. Native cellulose molecules appear predominantly as crystalline cellulose, which shows a high degree of intermolecular hydrogen bonding explaining its remarkable stability and recalcitrance to enzymes. Thus disrupting crystal intermolecular hydrogen bonds through cellulose swelling and dissolution with high-temperature operating cellulases overcomes recalcitrance and result in enzymatic digestion of native cellulose.

Results Isolation and Characterization of High-Temperature Operating and Thermostable Cellulases

A series of ten high-temperature operating and thermo-stable cellulases were identified through bioinformatics driven searches of archeal and bacterial genomes. The corresponding genes were genetically manipulated to adapt expression to a laboratory tractable system (Escherichia coli) by codon optimization and usage controlled promoters. Individual proteins were expressed and isolated (purified) from E. coli crude extracts and analyzed for activity and other physical and chemical properties. Data presented in tabular form in FIG. 9 describes the eight enzymes isolated in this study.

Termocel 1 and 2, group into a class with similar physical and catalytic properties, they exhibit a molecular weight of 34,005 and 31,930 D, a pI of 4.8 and 4.77 and a net charge at pH 7 of −13.10 and −13.30, respectively. They appear to function through an exo-processive-endoglucanase cleaving pattern with a specific activity on Avicel of 63.4 and 8.1 U and on swollen cellulose of 13.6 and 2.2. U, respectively.

Termocel 3 and 4 differ slightly with a molecular weight of 51,930 and 59,980 D, a pI of 6.47 and 7.05 and a net charge at pH 7 of −3.60 and 0.30, respectively. These enzymes also seem not to overlap with their predicted mode of operation, one exoprocessive type and the other as a endoglucanase with specific activity on Avicel of 48.5 and 6.8 and on swollen cellulose of 8.4 and 2.2 U, respectively.

Termocel 5 and 6 fall in a third class with similar physical and catalytic properties. They exhibit a molecular weight of 38,226 and 31,818 D, a pI of 5.58 and 5.66 and a net charge at pH 7 of −6.60 and −5.00, respectively. They appear to function through an internal cleaving pattern (endoglucanase) with a specific activity on Avicel of 34.1 and 20.6 U and on swollen cellulose of 6.8 and 5.1 U respectively.

Termocel 7 and 8 are β-glucosidases with distinct physical properties but similar catalytic activity. They exhibit a molecular weight of 81,243 and 51,509 D, a pI of 5.38 and 5.84 and a net charge at pH7 of −16.90 and −9.10, respectively. They cleave cellobiose with a specific activity on pNPG of 69.4 and 60.9 U, respectively.

Termocel 9 and 10 are endocellulases that exhibit a molecular weight of 45,059 and 85,598 D, a pI of 6.16 and 7.80 and a net charge at pH 7 of −2.20 and 4.30, respectively. They have a specific activity on Avicel of 5.2 and 4.9 U and on swollen cellulose of 1.4 and 1.5 U respectively.

Mode of Operation

FIGS. 10 and 11 describe the mode of operations of all six cellulases. Termocel 1, 2 and 3 are cellulases that function by sequentially cleaving glucose residues of the non-reducing end of a polymeric substrate. FIG. 10 shows the sequential depolymerization breakdown products through capillary zone electrophoresis.

Termocel 4, 5 and 6 are cellulases that function by internally cleaving a multimeric substrate. FIG. 11 shows trimeric and dimeric breakdown products, indicating internal cleavage of the pentameric substrate.

High-Temperature Catalytic Operation

FIG. 12 shows the optimum temperature of operation of eight Termocels in tabular form. The highest optimum was found for Termocel 1 with and optimum of 102° C. and the lowest optimum was found to be Termocel 6 with 85° C. At 60° C., all Termocels lost at least 40% of their activity (except Termocel 4) and at 20° C. the Termocels operated with less than 20% of their optimum activity.

Among the beta-glucosidases, no significant differences between activity and temperature optimum were apparent. However, catalytic inactivation at lower temperatures (45 and 20° C.) to levels below 1% residual activity for Termocel 7 is remarkable.

Thermal Stability

Thermostability of the Termocels was evaluated to determine the working time frame with useful enzymatic activity at high-temperatures. Enzymes were incubated at 90° C. for up to 5 days and than assayed for CMC (endo-glucanase and exo-cellulase) or PNPG (beta-glucosidase) activity and results are reported as % of residual activity in FIG. 13. With the exception of Termocel 3, all enzymes retained over 80% of their initial enzymatic activity after a 5-day incubation period at 90° C.

Modes of Use

These high-temperature operating cellulases can be used in all processes in which cellulose degradation at high temperatures is desired. These applications include but are not restricted to food processing, feedstuff preparation, textile finishing and paper pulping. The consolidated enzyme system is useful to hydrolyze fibrous crystalline cellulosic biomass materials, at high temperatures with Termocel 1, 2, 3, 4, 5, 6, 7 and 8 to produce high-sugar containing fermentation broths. In addition the genes of the high-temperature operating enzyme system can be used in producing transgenic organisms capable of expressing one or more high-temperature operating and thermostable plant cell wall degrading enzymes.

Methods Cloning

Genomic DNA of Pyrococcus horikoshii OT3 served as the PCR template for the amplification of the PH1171 gene. Likewise, genomic DNA of Thermotoga petrophila RKU-1 served as PCR template for the cloning of the PetroA, PetroB, Tpet_(—)0898 and Tpet_(—)0952 genes. Primer sequences are shown in Table 1. Restriction sites were introduced (bold letters). The O-eglA, ZP and E1 genes were synthesized without using a DNA template; the codons of the three genes were also optimized according to the sequences of corn and rice genomes (FIGS. 1A, 4A and 6A). All gene segments generated were cloned into the NcoI and XbaI sites of the pBAD/Myc-His vector (Invitrogen), which carries a fusion sequence (GAACAAAAACTCA TCTCAGAAG AGGATCTGAATAGCGCCGTCGACCATCATCATCATCATCATCAT, SEQ ID NO: 17) encoding six histidine residues at the C-terminus of any protein expressed from the vector (EQKLISEEDLNSAVDHHHHHH, SEQ ID NO: 18) The expression plasmids were used to transform Escherichia coli TOP 10F′ (Invitrogen). All constructs were verified by DNA sequencing.

TABLE 1  Oligonucleotide sequences used in this study. SEQ ID Primer Sequence (5′→3′)^(a) NO: Termocel 3 ATATCCATGGAGGGGAATACTATTCTTAAAATCGTACTAAT (Forward) 19 ATGCTCTAGAAACCTGGGAGCCCTTCTTAAG (Reverse) 20 Termocel 5 GAAACGCTCCTCCCTGTAGT (Forward) 21 ATGCTCTAGAAATTCTCTCACCTCCAGATCAATAGAGA (Reverse) 22 Termocel 2 AGGTGGGTAGTTCTTCTGATGG (Forward) 23 ATGCTCTAGAAATTTTACAACTTCGACGAAGAAGTCTTTGA (Reverse) 24 Termocel 7 ATATCCATGGGAAAGATCGATGAAATCCTTTCA (Forward) 25 ATGCTCTAGAAATGGTTTGAATCTCTTCTCTCCC (Reverse) 26 Termocel 8 AACGTGAAAAAGTTCCCTGAAG (Forward) 27 ATGCTCTAGAAAATCTTCCAGACTGTTGCTTTTG (Reverse) 28 ^(a)Boldface indicates sequences complementary to the primers used to amplify the selectable markers.

Expression and Purification

An overnight growth of transformed E. coli strain containing the fusion protein vector was inoculated into fresh Luria-Bertani medium containing ampicillin. When the OD⁶⁰⁰ reached 0.5-0.6, L-arabinose was added to a final concentration of 0.2%. The culture was allowed to grow for another 4-5 h at 37° C. and the cells were collected by centrifugation. The pellet was stored at −80° C. prior to further processes. Cells were disrupted by sonication and the cell debris was removed by centrifugation at 10,000×g for 20 min. The protein pool was then heat treated at 95° C. for 5 min, and denatured proteins were removed by centrifugation at 12, 000×g for 20 min. The recombinant protein carrying a His6 tag was then purified by immobilized metal-chelate affinity chromatography (Qiagen). Hydrolysis of cellulose, hemicellulose and starch Hydrolysis of Avicel PH101, carboxymethyl cellulose (CMC), xylan from birch wood, α-cellulose, β-glucan barley, laminarin, lichenan, starch, swollen Avicel PH101, wheat arabinoxylan, xylan from beechwood and xylan from oat-spelt was measured spectrophotometrically by the increase of reducing ends at various temperatures and pH. The amount of reducing sugar ends was determined by the dinitrosalicyclic acid (DNS) method. The assay mix contained 10 μl of diluted enzymes, 30 μl of 100 mm sodium phosphate buffer, pH 6.0, and 20 μl of 0.5% (wt/vol) soluble substrates or 1% slurries (wt/vol) of insoluble substrates for 30 min or 1 hour. The reaction was terminated by adding 60 μl of DNS Solution. The absorbance of assay mix was read at 575 nm after the incubation at 100° C. for 5 min. The activity of enzymes as a function of temperature and pH was measured with CMC. Temperature gradient was achieved using PCR cycler (MJ Research). Phosphate/citrate buffers were used to generate pH gradient (ie., 2, 3, 4, 5, 6, 7, 8, 9.1). For the thermostability assay, each enzyme was incubated at 90° C. An aliquot of enzymes was taken each day. Residual activity was measured with CMC.

Hydrolysis of p-Nitrophenol-β-D-Glucoside

Activity of β-glucosidase was determined spectrophotometrically by monitoring the release of p-nitrophenol from the substrate p-nitrophenol-β-D-glucoside (Sigma) at various temperatures and pH. The assay mix contained 10 μl of diluted enzymes, 30 μl of 100 mm pH buffer, and 20 μl of 50 mM p-nitrophenol-β-D-glucoside for 10 min. The reaction was terminated by adding 120 μl of 1M Na₂CO₃. The absorbance of assay mix was read at 412 nm. Temperature and pH dependent activities and thermostability were measured as described above except that p-nitrophenol-β-D-glucoside was used as substrate.

Capillary Electrophoresis of Oligosaccharides

Capillary electrophoresis of oligosaccharides was performed on a BioFocus 2000 (Bio-Rad Laboratories,) with laser-induced fluorescence detection. A fused-silica capillary (TSP050375, Polymicro Technologies) of internal diameter 50 μm and length 31 cm was used as the separation column for oligosaccharides. The samples were injected by application of 4.5 lbin-2 of helium pressure for 0.22 sec. Electrophoresis conditions were 15 kV/70-100 μA with the cathode at the inlet, 0.1 M sodium phosphate, pH 2.5, as running buffer, and a controlled temperature of 20° C. The capillary was rinsed with 1 M NaOH followed by running buffer with adip-cycle to prevent carryover after injection. Oligomers labeled with APTS were excited at 488 nm and emission was collected through a 520-nm band pass filter.

Biomass Substrate Specificity of Termocels

A table depicting the biomass substrate specificity of Termocels 1-8 is provided as FIG. 15.

While the invention has been described in terms of its preferred embodiments, those skilled in the art will recognize that the invention can be practiced with modification within the spirit and scope of the appended claims. Accordingly, the present invention should not be limited to the embodiments as described above, but should further include all modifications and equivalents thereof within the spirit and scope of the description provided herein. 

1-5. (canceled)
 6. A method of hydrolyzing cellulose, comprising the steps of contacting said cellulose with one or more recombinant cellulase enzymes with an amino acid sequence represented by an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ. ID NO: 30 and SEQ ID NO: 32; and activating said one or more recombinant cellulase enzymes by the application of heat.
 7. A method of hydrolyzing cellulose to produce glucose, comprising the step of heating said cellulose to a temperature in the range of 70° C. to 100° C. in the presence of one or more recombinant cellulase enzymes, wherein said one or more recombinant cellulase enzymes includes: at least one recombinant endoglucanase selected from the group consisting of Termocel 3, Termocel4, Termocel 5, Termocel 6, Termocel 9 and Termocel 10; at least one recombinant exoprocessive-endoglucanase selected from the group consisting of Termocel 1 and Termocel 2; and, at least one recombinant beta-glucosidase selected from the group consisting of Termocel 7 and Termocel
 8. 8. A method of fermenting glucose to produce ethanol, comprising the steps of providing a source of cellulose; heating said cellulose to a temperature in the range of 80° C. to 100° C. in the presence of at least one recombinant endoglucanase enzyme selected from the group consisting of Termocel 3, Termocel4, Termocel 5, Termocel 6, Termocel 9 and Termocel 10; at least one recombinant exoprocessive-endoglucanase enzyme selected from the group consisting of Termocel 1 and Termocel 2; and at least one recombinant beta-glucosidase enzyme selected from the group consisting of Termocel 7 and Termocel 8; wherein said step of heating is carried out under conditions whereby at least a portion of said cellulose is hydrolyzed to glucose; and fermenting said glucose to produce ethanol.
 9. The method of claim 8, wherein said step of fermenting is catalyzed by one or more yeasts.
 10. (canceled) 